DRUGS OBTAINED FROM MARINE SOURCES WITH SPECIAL REFERENCE TO ANTIMICROBIAL COMPOUNDS:

INTRODUCTION:

INTRODUCTION The phrase ‘drug molecules’ has been appropriately & judiciously employed in line with the classical concept,that it is a specific chemical entity which essentially possesses a marked & pronounced pharmacological activity emphatically on the mammalian organism. Marine Drugs :- The drug obtained from marine organisms which are being conventionally used like shark & cod-liver oils,sodium alginate,agar-agar,chitin etc. Importance of Marine Drugs :- Marine organisms are potential source for drug discovery.Life has originated from the oceans that cover over 70% of the surface of earth & contain highly ecological,chemical & biological diversity starting from micro-organisms to vertebrates.This diversity has been the source of unique chemical compounds,which hold tremendous pharmaceutical potential.

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Trends in drug discovery from natural sources emphasize on investigation of the marine ecosystem to explore numerous complex entities.These entities are the sources of new leads for treatment of many diseases such as cancer,aids,inflammatory conditions & a large variety of viral,bacterial & fungal diseases. Because of the highly chemical & physical harsh conditions in marine environment,the organisms produce a variety of molecules with unique structural features & exhibit various types of biological activities. Majority of the marine natural products have been isolated from sponges,coelenterates (sea whips,sea fans & soft corals), tunicates,opisthobranch echinoderms( starfish,sea cucumbers,etc ) & bryozoans & a wide variety of marine micro-organisms in their tissues.

ANTIMICROBIAL DRUGS FROM MARINE SOURCES:

ANTIMICROBIAL DRUGS FROM MARINE SOURCES A plethora of important antimicrobial drug substances have been isolated, characterised & studied extensively over the past three decades particularly from the vast domain of marine organisms. A few typical examples are :brown algae,red algae( viz three different species),gorgonian corals; & sponge( viz two variant species). The chemical substances from these species shall now be discussed briefly as under:-

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(1.) ZONAROL Biosource : Zonarol and Iso-zonarol are both obtained from Dictyopteris zonaroides (Brown algae). Use : Antifungal in nature.

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(2.)TETRABROMO-2-HEPTANONE Biosource : tetrabromo-2-heptanone is obtained from yet another species of Red algae Bonnemaisonia hemifera . (3.) 2-CYANO-4,5-DIBROMOPYRROLE It is perhaps one of the rarest examples of a chemical entity isolated from a marine organism which contains a cyano (-CN) function group. Biosource : It is obtained from Agelas oroides , a specific type of sponge found in marine sources.

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(4.) AEROPLYSININ -1(+) AND AEROPLYSININ -1(-) Biosource : The two isomers,viz Aeroplysinin -1(+) and Aeroplysinin -1(-) are obtained from Verongia aerophoba , another species of sponge. (5.) EUNICIN Biosource : Eunicin is obtained from Eunicin mammosa the well known Gorgonian corals .

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(6.) CEPHALOSPORIN-C(26) Cephalosporin-c is the semi-synthetic derivative of Cephalothin sodium;Use : It is widely used as an antibiotic drug. (7.) ISTAMYCIN A (27) & (28) Produced by fermentation of the marine Streptomyces B.S- Streptomycete-Streptomyces tenjimariensis ss-939(in vitro activity against gram-(- ve ) & + ve bacteria;Use:Antibacterial,effective on Aminoglycoside resistant bacteria,stachybtrin A & B isolated from a brakish water fungus,inhibit antimicrobial activity. (8.)ACANTHELIN Org. is Acanthella acuta;Active against mycobacterium.

EXPERIMENTAL APPROACHES FOR EVALUATION OF ANTIMICROBIAL ACTIVITY :-:

EXPERIMENTAL APPROACHES FOR EVALUATION OF ANTIMICROBIAL ACTIVITY :- IN VITRO ANTIMICROBIAL SUSCEPTIBILITY TESTING - The antimicrobial susceptibility test(AST) is an essential technique used in pathology to determine resistance of certain microbial strains to different antimicrobials & in pharmacology research it is used to determine the efficacy of novel antimicrobials from biological extract against different micro- organisms.These methods are widely employed to determine Minimum Inhibitory Concentration of the antimicrobial substance.AST standard tests are classified into diffusion & dilution methods.Diffusion tests are agar well diffusion,agar disk diffusion,poison food technique & bioautography,while dilution methods are agar dilution,broth microdilution & broth macrodilution technique. AGAR DISK DIFFUSION ASSAY - This antimicrobial test was developed in 1940.In this method 6mm sterilised filter papers disks( whatmann no.1) are saturated with filter sterilised plant extract of desired concentration.The impregnated discs are then placed onto the surface of a suitable solid agar

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medium like Mueller Hinton,Trypton soy agar or Nutrient agar.The media has been pre- innoculated with test organisms.The std inoculum size is of 1 x 108 cfu /ml of bacteria for inoculating diffusion plates.The drying time of impregnated paper disk varies from 2h to overnight under a laminar flow cabinet.Plates are then incubated for 24h at 37 degree celsius (bacteria) & 48h at 25 degree celsius (fungi).After incubation zone diameter is measured to the nearest whole millimeter at the point wherein there is a prominent reduction of 80% growth. AGAR WELL DIFFUSION ASSAY - The principle is same as that of agar disk diffusion assay.A standardised concentration of inoculum with fixed volume is spread evenly on the surface of gelled agar plate.A hole which ranges from 6-8mm in diameter is punched with a sterile cork borer aseptically in the middle.A fixed volume of plant extract is then introduced into the bored agar well & incubated at optimum temperature & duration depending upon the test micro-organism.

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POISON FOOD TECHNIQUE - Generally antifungal activity is determined by this technique.5 days old fungal culture is punched aseptically with a sterile borer of generally 7mm diameter.The fungal discs are then put on the gelled agar plate.The agar plates have been prepared by impregnating desired concentration of plant extract at a temperature of 45-50 degree celsius.The plates are then incubated at 26 + 1 degree celsius for fungi.Colony diameter is recorded by measuring the two opposite circumference of the colony growth. % Mycelial growth=< Mycelial growth(control)- Mycelial growth(treatment)/ Mycelial growth(treatment)>x100 SPORE GERMINATION ASSAY - Plant extract of desired concentration & volume are added to the surface of dried slides as a film or in a cavity of a cavity slide.Fixed volume & std concentration of spore suspension of test fungi are spread over the film whereas in controlled treatment,

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Distilled water is added in place of spore suspension.Slides are then placed on a glass rod in petridish under moistened conditions & incubated for 24 degree celsius.After incubation,slides are fixed in lacto phenol cotton blue & observed microscopically for spore germination. %Spore germination=<Germinated spores(no.)/Total spores(no.)>x100 BROTH MICRODILUTION - The microtitre plate or broth microdilution method has provided potentially useful technique for determining Maximum Inhibitory Concentration(MIC) of large nos. of test samples.MIC in microbiology is the lowest concentration of antimicrobials that will inhibit the visible growth of micro-organisms after overnight incubation.A stock solution of the extract is 1 st obtained in solvent.Mueller Hinton broth or water is often used as a diluent in

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the wells of the microtitre plate before transferring an equal volume of stock solution to the plate.Two fold serial dilutions are made from the 1 st well to obtain a concentration range.MIC-5-8 concentration can represent achievable concentrations for the used antimicrobials.The inoculum size for this procedure is usually 1 x 10^6 CFU/ ml.An equal volume of microbial culture is added to the wells & incubated at 37 degree celsius for 24h.After incubation, the plates are examined for changes in turbidity as an indicator of growth.The 1 st well that appears clear is taken to be the MIC of the extract.Indicators like tetrazolium salts or resazurin dye or spectrophotometry can be used to determine the presence of growth.For spectrophotometric method the absorbance is usually at 620nm with negative control as blank.Concentration with sharp decline in absorbance value is the MIC of the test phytochemical .

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BROTH MACRODILUTION ASSAY - Here the basic principle is same as the broth microdilution assay,but the test is performed in a test tube.A set of test tubes with different concentrations of extracts with the same volume are prepared.Tubes are inoculated with test micro-organisms of std concentrations.After incubation, tubes are examined for changes in turbidity as an indicator of growth.MIC of the test phytochemical can be determined using the above discussed methods. BIOAUTOGRAPHY - It is a very convenient way of testing plant extracts & pure phytochemical compounds for their effect on both human pathogenic & plant pathogenic micro- organisms.It is employed in the target directed isolation of active constituents,as a preliminary phytochemical screening technique, by bioassay guided fractionation,

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to detect active components.Bioautography methods are usually grouped into three categories, agar diffusion or contact bioautography,immersion or agar overlay bioautography & direct bioautography . AGAR DIFFUSION OR CONTACT BIOAUTOGRAPHY - Here, antimicrobials are diffused from a thin layer chromatographic plate(T.L.C) to an inoculated agar plate.The chromatogram is placed onto the inoculated agar layer & left for sometime to enable diffusion.Then the chromatogram is removed & the agar layer is incubated.The inhibition zones are observed on the agar surface in places where the spots of antimicrobials are stuck to the agar.The method resembles a disk assay.

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IMMERSION OR AGAR OVERLAY BIOAUTOGRAPHY - Here the chromatogram is covered with a molten,seeded agar medium.After solidification,incubation & staining(usually with tetrazolium dye), the inhibition or growth bands are visualized. DIRECT BIOAUTOGRAPHY - A determined amount of plant extract is applied to silica 60 gel plates & developed with an appropriate solvent system to separate the phytochemicals.A suspension of test bacteria is sprayed onto the T.L.C plate.The bioautogram is then incubated at 25 degree celsius for 48h in humid conditions.For visualization of microbial growth,tetrazolium salts are used,which are converted by the dehydrogenases of living micro-organisms to intensely coloured formazen.These salts are sprayed onto the bioautogram & are re-incubated at 25 degree celsius for 24h or at 37 degree celsius for 3-4h.

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Clear whites zones against a purple background on the T.L.C plate indicate antimicrobial activity of the extract. One good example is the Antimicrobial activity evaluation of the oleoresin oil of Pistacia vera L. It is a member of the Anacardiaceae family.The essential oil was extracted from resin by hydrodistillation with ethanol using a Clevenger apparatus.The combined hydroalcoholic extract was filtered through a filter paper & evaporated to dryness under reduced pressure in a Rotavapor & then stored in the dark at 4 degree celsius in an air tight container. Three bacterial strains viz. E.coli,Proteus spp & S. aureus are used. DETERMINATION OF ANTIMICROBIAL ACTIVITY : 3 methods were used to determine the activity; agar disk diffusion method,determination of MIC & dilution broth method.

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The agar disk diffusion method was employed to determine the antimicrobial activities of the essential oil.It was a simple,cheap & reproducible practical method.A suspension of each tested micro-organism diluted prior to 10^-1,10^-2 & 10^-3-(1 ml of 10^8 cells/ml) was spread on a solid agar medium in Petri dishes(Mueller Hinton agar).Filter paper disks (6 mm in diameter) were soaked in 13 microliter of the resin oil & placed on the inoculated plates & allowed to dry for 15 mins,then incubated at 37 degree celsius for 24H.The diameters of the inhibition zones were measured in millimeters. MIC was taken from the concentration of the lowest dosed test tube showing visually no growth after 24h.10 microliter from each visually ungrown test tube were subcultured on a Mueller Hinton agar.S . aureus,Proteus spp. & E.coli were diluted at 10^-1,10^-2,10^-3; a control

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is prepared with ethanol & oleo resin of Pistacia vera.Each dilution of the slick strain is spread over the surface of the Petri dish containing the Mueller Hinton agar medium liquid which must be dried at 37 degree celsius for 15 mins . Then 4 disks were placed on an agar containing 0.5,1.5,2 & 2.5 microliter of the oleoresin oil dilution.The control disk is impregnated with ethanol & placed in the petridish center,to be incubated towards the end at 37 degree celsius for 24h. Dilution broth susceptibility assay was used for the antimicrobial evaluation.Stock soltns of the resin oils were prepared in ethanol by mixing 1 ml of the extracts with 9 ml of alcohol in test tubes to obtain the mother soltns,followed by succesive dilutions at 10^-2 & 10^-3.The control was prepared by mixing 1 ml of distilled water with 9 ml of

SOME OF THE MARKETED DRUGS:-:

SOME OF THE MARKETED DRUGS:-

FUTURE PROSPECTS:-:

FUTURE PROSPECTS:- Marine natural product research has just started to bloom.Today marine sources have the highest probability of yielding natural products with unprecendented carbon skeletons & interesting biological activity. Many more prospects regarding new habitats,e.g black smokers,deep ocean samples & Antartic areas are still wide open for research. The vast area of research into marine micro-organisms comprising of marine bacteria,ranging from archaebacteria to gliding bacteria,fungi & whale,range of microalgaes,e.g dinofla yellates,diatoms,protozoa,e.g ciliaks,amoebas flagellates is just emerging & showing immense potential.

CONCLUSION :

CONCLUSION In short,the marine-world evidently & explicitly enjoys status of holding an enormous & tremendous potent towards epoch making discovery of a plethora of altogather newer Lead Molecules in the development of medicinally potent therapeutic agents that are active against a variety of parasites & infectious ailments. Inorder to explore,tap & above all exploit commercially the relatively virgin biological reserves exclusively depends on the use of rapid technological advancement towards their collection,preservation,identification & characterization of trace quantum of essential secondary metabolites.With the advent of recent development & use of technologically advanced computer aided sophisticated analytical instruments have turned this novel dream like fiction into reality.